| Abstract | Ateroskleroza je kronična fibro-proliferativna i imuno-inflamatorna bolest arterija, karatkerizirana endotelnom disfunkcijom, lokalnom oksidacijom cirkulirajućih lipoproteina, njihovom akumulacijom u stijenku i aktivacijom gena za proinflamatorne citokine. Klinička prezentacija ateroskleroze nastaje uslijed destabilizacije plaka pod djelovanjem lokaliziranog upalnog procesa. EL je nova članica skupine lipaza koja zbog svoje povezanosti s upalnim procesom može imati važnu ulogu u destabilizaciji plaka. Njena ekspresija povećana je pod utjecajem upalnih citokina, oksidiranog LDL-a i lipopolisaharida i to u makrofazima, ključnom celularnom elementu destabilizacije plaka. S druge strane, EL ima adhezivnu ulogu za monocite te svojom lipolitičkom aktivnošću oslobađa slobodne masne kiseline i lizo-fosfatidilkolin koji aktiviraju upalne citokine. Osnovni cilj ovog istraživanja bio je istražiti povezanost ekspresije EL u karotidnom plaku i plazmatske razine EL s kliničkom slikom bolesnika i s histološkim izgledom plaka. U istraživanje je uključeno 66 bolesnika sa značajnom (>70%) unilateralnom stenozom karotidne arterije hospitaliziranih radi karotidne endarterektomije. Bolesnici su podijeljeni na dvije osnovne skupine: 1) asimptomatski bolesnici (n=42) i 2) simptomatski (n=24). Karotidna stenoza smatrana je simptomatskom ako je bolesnik imao žarišni neurološki simptom (CVI, TIA ili amaurosis fugax) unutar perioda od 4 mjeseca koji po karakteru pripadaju području ciljne karotide. Na temelju histološke analize plakovi su prema svojim značajkama nestabilnosti podijeljeni u tri skupine: 1) fibrozni (F) prekriveni glatkom luminalnom površinom, 2) ulcerirani – nekomplicirani (UNC) s ulceriranom i tankom fibroznom kapom i 3) ulcerirani-komplicirani (UC) s ulceriranom i tankom fibroznom kapomm, krvarenjem unutar plaka i/ili trombom. Imunhistokemija je učinjena sa zečjim klonskim anti-humanim protutijelim na CD163 (specifičnim za makrofage) i sa zečjim anti-EL protitijelima. Ekspresija EL izražena je semikvantitativno kao: (A) slabije bojanje (0–25% stanica pozitivno) i (B) jače bojanje (>25% stanica pozitivno). Plazmatska razina EL određena je ELISA metodom u 55 bolesnika.
----- Rezultati: Simptomatski bolesnici češće su imali visoko-signifikantnu (95-99%) stenozu (p=0,027) te hipoehogene (meke) zone u plaku (p=0,039), međutim nije bilo značajne razlike u histološkoj slici (p=0,213). U F plakovima imunohistokemija za EL bila je slaba (tip A) i smještena u endotelnom sloju i u stanicama unutar fibrozne kape. Kod UC plakova imunohistokemija za EL bila je jača (tip B) i smještena između nekrotične srži i fibrozne kape gdje su se dominantno nalazili makrofazi. Jače EL imunobojanje (tip B) bilo je češće kod UC plakova nego kod UNC, a osobito F plakova (79:50:33%, p=0,004). Plazmatska razina EL bila je izraženija kod hipertoničara (p=0,004) i pušaća (p=0,058) te kod bolesnika liječenih ACE inhibitorima (p=0,018) i kalcijskim blokatorima (p=0,05). Nije bilo povezanosti između ekspresije EL u plaku i plazmatske razine EL. Korelacijskim testom nije nađena značajna povezanost plazmatske razine EL s hsCRP-om, IL6, HDL-om i LDL-om. U usporedbi sa skupinom bez simptoma skupine svježi simptomi (simptomi unutar 4 mjeseca i sa lokalizacijskom povezanošću s ciljnom karotidnom arterijom) i stari simptomi (preostali simptomatski bolesnici) imali su značajno veću plazmatsku razinu EL (p=0,011), dok između te dvije skupine simptomatskih bolesnika nije bilo razlike (p=0,459).
Potvrđena hipoteza da je EL jače izražena u karotidnim plakovima simptomatskih bolesnika i u histološki vulnerabilnim plakovima ukazuje na njenu povezanost s procesom destabilizacije plaka. Tu povezanost dodatno učvršćuje i činjenica da je EL u aterosklerotskom plaku najviše izražena između fibrozne kape i nekrotične srži odnosno na mjestu gdje su najzastupljeniji makrofazi. U ovom radu nije nađena povezanost između histološke slike plaka i simptoma što je prisutno i u ranijim istraživanjima i ukazuje na nedostatak dovoljno specifičnih i senzitivnih kriterija kojima bi se mogla procjeniti vulnerabilnost plaka. Značajna povezanost EL i s histološkom slikom plaka i sa simptomatologijom upućuje da bi se EL potencijalno mogla koristiti kao jedan od histoloških markera vulnerabilnosti plaka. Plazmatska EL bila je povišena kod simptomatskih bolesnika bez obzira na vrijeme i lokalizaciju simptoma, a nije bila povezana s ekspresijom EL u plaku. Navedeno ukazuje da na razinu plazmatske EL ne utječe povećana ekspresija na jedom lokusu već ona moguće ovisi o globalnoj endotelnoj disfunkciji i općenito pojačanoj ekspresiji u svim arterijama. Pušenje i arterijska hipertenzija, čimbenici koji oštećuju endotel na kemijski i mehanički način i na taj način dovode do endotelne disfunkcije, bili su pozitivno povezani s plazmatskom razinom EL. HsCRP i IL-6 bili su iznad razine nađene kod normalne populacije, ali nije bilo očekivane povezanosti s kliničkom prezentacijom što je vjerovatno uzrokovano činjenicom da svi bolesnici već imaju uznapredovalu aterosklerozu. To implicira da je u toj fazi bolesti EL u plazmi osjetljiviji pokazatelj rizika za cerebrovaskularne (a možda i za kardiovaskularne) događaje nego hsCRP. |
| Abstract (english) | Atherosclerosis is a chronic fibro-proliferative and immuno-inflammatory artery disease characterized by the endothelial dysfunction, local oxidation of circulating lipoproteins, their accumulation in the vascular wall and activation of proinflammatory cytokine genes. Clinical presentation of atherosclerosis is initiated by plaque destabilization due to a local inflammatory process. EL is a new member of triglyceride lipase gene family which is likely to have an important role in plaque destabilization by way of local inflammation. EL expression is increased under the influence of proinflammatory cytokines, oxidized LDL and lipopolisacharides in essential cellular agents of plaque destabilization, macrophages. On the other hand, EL promotes monocytes/macrophages adhesion and cleaves HDL-phosphatidylcholine (HDL-PC) into bioactive molecules, lysophosphatidylcholines (lyso-PC) and free fatty acids (FFA) capable to activate inflammatory cytokines, via its phospholipase A1 activity. The main objective of this study was to evaluate the relationship between the following: the expression of EL in human carotid atherosclerotic plaque and serum level of EL versus clinical presentation and degree of plaque instability. Final study population included 66 patients with proven significant (>70%) unilateral stenosis of carotid artery admitted for carotid endarterectomy (CEA). Patients were grouped as follows: 1) asymptomatic group (42 pts) and 2) symptomatic group (24 pts). The carotid stenosis was considered symptomatic if a patient had suffered a focal neurological symptom (ipsilateral stroke, transient ischemic attack or monocular blindness), sudden in onset and referable to the respective carotid artery within 4 months of CEA. Histological examination was performed in order to find features indicative of plaque vulnerability. Plaques were defined as: 1) fibrous (F) covered by a smooth luminal surface, 2) ulcerated non-complicated (UNC) with thin, fissured or ruptured cap, and 3) ulcerated-complicated (UC) with thin fissured or ruptured cap, intra plaque hemorrhage and/or thrombus. Immunohistochemistry was performed using the rabbit clonal anti-human CD163 (specific for macrophages) and rabbit anti-EL antibody. The expression of EL was evaluated on a semi-quantitative scale as follows: 1) (A) low staining (0–25% positive cells), and 2) (B) high staining (>25% positive cells). EL plasma protein levels were analyzed in 55 patients by ELISA method.
----- Results: Symptomatic patients more frequently had highly significant (95-99%) stenosis (p=0,027) and echolucent zones in plaques (p=0,039), but there was no significant difference between the groups in histological features (p=0,213). In F plaques the EL immunostaining was weak (typ A) and detected in endothelial layer and in cells within the fibrous cap. In contrary, in UC plaques the EL immunostaining was more pronounced (type B) and localized between the necrotic core and the fibrous cap where macrophages dominated. The intensity of the EL immunostaining type B was most frequently observed in UC plaques, followed by the UNC and F plaques (79:50:33%, p=0,004). EL plasma levels were significantly higher in patients with arterial hypertension (p=0,004), smokers (p=0,058), patients taking ACE inhibitors (p=0,018) and Ca blockers (p=0,05). EL plasma levels were similar in patients with the EL immunostaining A and B (p= 0,378) and were not significantly correlated with hsCRP, IL-6, HDL and LDL plasma levels. Compared to the no symptoms group the new symptoms group (symptoms present within 4 month referable to carotid artery) and the old symptoms group (other symptomatic patients) had significantly higher EL plasma level (p=0,011) whereas the two symptomatic groups of patients showed no significant difference.
The confirmation of hypothesis that the EL is more expressed in carotid plaques of symptomatic patients and in vulnerable plaques proves its connection with plaque destabilization. This connection is also supported by the fact that EL is mostly expressed in region between the necrotic core and the fibrous cap of vulnerable plaques where macrophages dominate. In line with some previous studies, there was no correlation between histological type and symptoms, which suggests the lack of sensitive and specific histological criteria to confirm plaque vulnerability. The significant relationship between the EL immunostaining and histologic features as well as the EL immunostaining and symptoms suggests that EL could be used in future as one of the markers of plaque vulnerability. EL plasma levels were increased in symptomatic patients irrespective to time of occurrence and localization of symptoms. Furtermore, EL plasma levels were not associated with the intensity of EL immunostaing within carotid plaques. This suggests that EL plasma level is not significantly influenced by increased focal expression of the EL in one plaque. Rather it is influenced by global endothelial disfunction and generally increased expression in all arteries. Higher levels of EL plasma levels were found in patients with arterial hypertension and smoking as two risk factors capable of inducing endothel disfunction, the former mechanically and the latter chemically. HsCRP and IL-6 levels where higher than in healthy population, but there was noconnection to symptoms, probably due to the advanced stage of atherosclerosis. This implicates that EL plasma level is at this stage of disease probably a more sensitive marker of cerebrovascular (and possible cardiovascular) events than hsCRP. |
